The proposed project seeks to further characterize a unique class of cell-penetrating peptides (CPPs) capable of translocating through the cell membrane independent of endocytosis and without compromising the physiology of the cell membrane. We hypothesize that CPPs with these distinct features, referred to as membrane translocating peptides (MTPs), can be engineered to facilitate cell-impermeable therapeutics directly (spontaneously) into cells. In order to establish an efficient peptide based delivery platform must take into account the mechanism of cellular entry. Two possible mechanisms of cellular entry exist for CPP- conjugates; energy-dependent processes involving endocytosis or energy-independent processes involving spontaneous direct translocation. Utilizing endocytosis for cellular entry poses problems of endosomal sequestration and degradation of CPP-conjugates. Therefore, our engineering goals for MTPs and MTP- conjugates are to ensure that the cellular uptake mechanism relies upon spontaneous translocation. The specific aims of this project will identify and optimize MTP sequences for spontaneous translocation followed by assessing the limitations of MTPs to deliver a range of cell-impermeable cargoes into cells. Broader implications from this project will serve to expand what is currently considered feasible to improve the bioavailability of potential therapeutics once associated with MTPs. Drug design strategies could also improve with associations with MTPs by considering potential drug candidates once overlooked for being cell-impermeable.